A: The retention time of the major peak for L-lysine in the chromatogram of the Test preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the test for Content of L-lysine.

B: The retention time of the major peak in ceforanide in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparationas obtained in the Assay.

Mobile phase— Mix 62volumes of methanol and 38volumes of water,and adjust with glacial acetic acid to a pHof 3.0,making adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Stock standard solution— Transfer about 36mg of L-lysine,accurately weighed,to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Standard preparation— Transfer 2.0mLof Stock standard solutionto a glass-stoppered,10-mLvolumetric flask,add 2.0mLof a 1.4%solution of tris(hydroxymethyl)aminomethane and 3.0mLof a 1.5%solution of 1-fluoro-2,4-dinitrobenzene in dehydrated alcohol,insert the stopper tightly,and mix.Heat at 50in a water bath for 30minutes.Remove the flask from the water bath,allow to cool,dilute with methanol to volume,and mix.

Test preparation— Transfer about 150mg of Ceforanide for Injection,accurately weighed,to a 100-mLvolumetric flask,add water to volume,and mix.Transfer 2.0mLof the resulting solution to a glass-stoppered,10-mLvolumetric flask,and proceed as directed under Standard preparation,beginning with “add 2.0mLof a 1.4%solution of tris(hydroxymethyl)aminomethane.”

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains 5-to 10-µm packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the column efficiency determined from the derivatized L-lysine peak is not less than 1500theoretical plates,the tailing factor for the same peak is not more than 1.3,the resolution,R,between the derivatized L-lysine peak and the 1-fluoro-2,4-dinitrobenzene peak is not less than 4.5,the capacity factor,k¢,is not less than 4and not more than 6,and the relative standard deviation for replicate injections is not more than 1.5%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of L-lysine in the Ceforanide for Injection taken by the formula: in which Cis the concentration,in µg per mL,of L-lysine in the Stock standard solution,Mis the quantity,in mg,of Ceforanide for Injection taken,and rUand rSare the peak responses obtained from the Test preparationand the Standard preparation,respectively.Use this percentage to calculate,on an L-lysine-free basis,the result from Assay preparation 1obtained as directed in the Assay.

Mobile phase ,Standard preparation,and Chromatographic system—Prepare as directed in the Assayunder Ceforanide.

Assay preparation 1— Dissolve a suitable quantity of Ceforanide for Injection,accurately weighed,in Mobile phase,and dilute quantitatively and stepwise with Mobile phaseto obtain a solution having a concentration of about 1mg of ceforanide per mL.Use this solution within 5minutes.

Assay preparation 2 (where it is represented as being in a single-dose container)—Constitute Ceforanide for Injection in a volume of water,accurately measured,corresponding to the volume of solvent specified in the labeling.Withdraw all of the withdrawable contents,using a suitable hypodermic needle and syringe,and dilute quantitatively and stepwise with Mobile phaseto obtain a solution containing about 1mg of ceforanide per mL.Use this solution within 5minutes.

Assay preparation 3 (where the label states the quantity of ceforanide in a given volume of constituted solution)—Constitute Ceforanide for Injection in a volume of water,accurately measured,corresponding to the volume of solvent specified in the labeling.Dilute an accurately measured volume of the constituted solution quantitatively and stepwise with Mobile phaseto obtain a solution containing about 1mg of ceforanide per mL.Use this solution within 5minutes.

Procedure— Proceed as directed for Procedurein the Assayunder Ceforanide.Calculate the quantity,in µg,of ceforanide (C20H21N7O6S2)in each mg of the Ceforanide for Injection taken by the formula: in which Mis the concentration,in mg per mL,of Assay preparation 1based on the weight of Ceforanide for Injection taken and the extent of dilution,and the other terms are as defined therein.Calculate the quantity,in mg,of C20H21N7O6S2withdrawn from the container,or in the portion of constituted solution taken by the formula: in which Lis the labeled quantity,in mg,of ceforanide in the container,or in the volume of constituted solution taken,Dis the concentration,in mg of ceforanide per mL,of Assay preparation 2or Assay preparation 3,based on the labeled quantity in the container or in the portion of constituted solution taken,respectively,and the extent of dilution,and the other terms are as defined therein.